Each year, over 30,000 patients suffering from hematological diseases such as leukemia, lymphoma, multiple myeloma, and severe aplastic anemia are treated with high-dose chemotherapy followed by hematopoietic stem cell (HSC) transplantations. The ability of HSCs to reconstitute all blood lineages post transplantation can be life-saving for these patients. However, their need is severely outweighed by their availability due to the difficulty in finding a suitable donor match coupled with their low expansion potential ex-vivo. A variety of culture conditions have been evaluated for in vitro expansion of HSCs but have yielded limited success due to an increase in differentiation, loss of stemness and promotion of cell cycle abnormalities in culture. Thus, this project aims to develop novel biomimetic biomaterial substrates and harbor the innate ability of co-existing niche cells to promote HSC expansion in vitro. Aim 1 will develop a biomaterial substrate to emulate the native bone marrow extracellular matrix environment and promote HSC expansion in vitro. Aim 2 will investigate if alternative sources of Nestin positive MSCs exist, which can be harnessed towards the expansion of HSCs in vitro. Finally, Aim 3 will determine the role of additional candidate niche cells in promoting HSC expansion in vitro. This proposal will characterize the molecular mechanisms behind HSC expansion due to the effects of the biomaterial or other candidate niche cells in culture with HSCs by analyzing cell surface receptors, cytoskeleton changes, soluble factors, genetic changes and associated intracellular pathways. Innovative methods for promoting HSC expansion could be used for life-saving transplantations to significantly impact the prognosis of patients with hematological malignancies. Additionally, this system can serve as a model for studying niche cell-cell interactions, cell-matrix interactions, drug screening and genetic diseases. These studies will significantly contribute to the knowledge base of the bone marrow niche microenvironment and potentially lead to the development of new patient therapies so they can live longer and more fulfilling lives.